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J. Biol. Chem., Vol. 282, Issue 12, 9182-9194, March 23, 2007

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Structural Characterization of Human 8-Oxoguanine DNA Glycosylase Variants Bearing Active Site Mutations^*

Christopher T. Radom¹, Anirban Banerjee², and Gregory L. Verdine^¶3

From the Departments of Chemistry and Chemical Biology and Molecular and Cellular Biology, Harvard University, Cambridge, Massachusetts 02138 and ^¶Program in Cancer Chemical Biology, Dana-Farber Cancer Institute, Boston, Massachusetts 02115

The human 8-oxoguanine DNA glycosylase (hOGG1) protein is responsible for initiating base excision DNA repair of the endogenous mutagen 8-oxoguanine. Like nearly all DNA glycosylases, hOGG1 extrudes its substrate from the DNA helix and inserts it into an extrahelical enzyme active site pocket lined with residues that participate in lesion recognition and catalysis. Structural analysis has been performed on mutant versions of hOGG1 having changes in catalytic residues but not on variants having altered 7,8-dihydro-8-oxoguanine (oxoG) contact residues. Here we report high resolution structural analysis of such recognition variants. We found that Ala substitution at residues that contact the phosphate 5' to the lesion (H270A mutation) and its Watson-Crick face (Q315A mutation) simply removed key functionality from the contact interface but otherwise had no effect on structure. Ala substitution at the only residue making an oxoG-specific contact (G42A mutation) introduced torsional stress into the DNA contact surface of hOGG1, but this was overcome by local interactions within the folded protein, indicating that this oxoG recognition motif is "hardwired." Introduction of a side chain intended to sterically obstruct the active site pocket (Q315F mutation) led to two different structures, one of which (Q315F^*149) has the oxoG lesion in an exosite flanking the active site and the other of which (Q315F^*292) has the oxoG inserted nearly completely into the lesion recognition pocket. The latter structure offers a view of the latest stage in the base extrusion pathway yet observed, and its lack of catalytic activity demonstrates that the transition state for displacement of the lesion base is geometrically demanding.

Received for publication, September 21, 2006 , and in revised form, November 13, 2006.

The atomic coordinates and structure factors (code 2NOB (H270A'), 2NOE (G42A), 2NOI (G42A'), 2NOH (Q315A), 2NOF (Q315F^*149), 2NOL (LRC^*292), and 2NOZ (Q315F^*292)) have been deposited in the Protein Data Bank, Research Collaboratory for Structural Bioinformatics, Rutgers University, New Brunswick, NJ (http://www.rcsb.org/).

^* This project was supported in part by National Institutes of Health Grant CA100742. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The on-line version of this article (available at http://www.jbc.org) contains a supplemental table and Figs. 1-9.

¹ Supported by a National Institutes of Health training grant.

² Present address: Rockefeller University, New York, NY 10021.

³ To whom correspondence should be addressed. Tel.: 617-495-5323; Fax: 617-495-8755; E-mail: gregory_verdine{at}harvard.edu.

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